Impact of malpractice liability among spine surgeons: A national survey of French private neurosurgeons.

PURPOSE: In the general context of medical judicialization, spine surgeons are impacted by the part that medical responsibility and the risk of malpractice play in their actions and decisions. Our aim was to evaluate possible shifts in practices among private neurosurgeons who are highly exposed to this judicial risk and detect alterations in their pleasure in exercising their profession. We present the first national survey on French physicians' perception of surgical judicialization and consequences on their practice. METHODS: An online survey was submitted to the 121 members of the French Society of Private Neurosurgery, who represent 29.1% of the total number of spine surgeons and perform 36.0% of the national total spine surgery activity. The French law (no-fault out-of-court scheme) significantly impacts these surgeons in the event of litigation. RESULTS: A total of 78 surveys were completed (64.5% response rate): 89.7% of respondents experienced alteration of doctor-patient relationship related to judicialization and 60.2% had already refused to perform risky surgeries. Fear of being sued added negative pressure during surgery for 55.1% of respondents and 37.2% of them had already considered stopping their practice because of this litigation context. CONCLUSION: The increasing impact of medical liability is prompting practitioners to change their practice and perceptions. The doctor-patient relationship appears to be altered, negative pressure is placed on physicians and defensively, some neurosurgeons may refuse high-risk patients and procedures. This situation causes professional disenchantment and can ultimately prove disadvantageous for both doctors and patients.

[Effectiveness of adjuvant corticosteroid therapy for chronic subdural hematoma: a retrospective study of 198 cases]. / Efficacité de la corticothérapie dans le traitement adjuvant des hématomes sous-duraux chroniques. Etude rétrospective sur 198 cas.

BACKGROUND AND PURPOSE: Adjuvant treatments can be proposed in addition to surgery for patients with chronic subdural hematoma (CSDH) in order to improve the postoperative outcome. According to the survey published in 2001 by the Neurosurgery French Society, 38% of French neurosurgeons use adjuvant corticosteroid therapy after surgery. Does this adjuvant corticosteroid therapy have an effect on the postoperative outcome of CSDH? METHODS: A retrospective trial was performed on patients who were surgically treated for CSDH between January 1998 and July 2002 in the Nice Department of Neurosurgery. Corticosteroid therapy was initiated just after surgery and maintained for one month. Part of the patients were not given corticosteroids enabling a comparison of two groups: "corticosteroid therapy" versus "no corticosteroid therapy". RESULTS: One hundred and ninety-eight patients were included in the trial, 142 patients in the "corticosteroid therapy" group and 56 patients in the "no corticosteroid therapy" group. The difference in survival between the two groups was significant in favor of the group give corticosteroids. A multivariate analysis was carried out which confirmed the beneficial effect of the corticosteroid therapy on survival of the operated patients. Their risk of death was threefold less than those not given this treatment (p=0.006). CONCLUSIONS: This study highlighted a protective effect of postoperative corticosteroid therapy on patient survival. This effect persisted at multivariate analysis. However, due to skews inherent in retrospective studies, a multicentric prospective randomized trial is being prepared in our institution to confirm these results.

Relevance of neutrophils in the murine model of haemolytic uraemic syndrome: mechanisms involved in Shiga toxin type 2-induced neutrophilia.

It has been demonstrated that infections due to Shiga toxins (Stx) producing Escherichia coli are the main cause of the haemolytic uraemic syndrome (HUS). However, the contribution of the inflammatory response in the pathogenesis of the disease has also been well established. Neutrophils (PMN) represent a central component of inflammation during infections, and patients with high peripheral PMN counts at presentation have a poor prognosis. The mouse model of HUS, by intravenous injection of pure Stx type 2 (Stx2), reproduces human neutrophilia and allows the study of early events in the course of Stx2-induced pathophysiological mechanisms. The aim of this study was to address the contribution of PMN on Stx2 toxicity in a murine model of HUS, by evaluating the survival and renal damage in mice in which the granulocytic population was depleted. We found that the absence of PMN reduced Stx2-induced lethal effects and renal damage. We also investigated the mechanisms underlying Stx2-induced neutrophilia, studying the influence of Stx2 on myelopoyesis, on the emergence of cells from the bone marrow and on the in vivo migration into tissues. Stx2 administration led to an accelerated release of bone marrow cells, which egress at an earlier stage of maturation, together with an increase in the proliferation of myeloid progenitors. Moreover, Stx2-treated mice exhibited a lower migratory capacity to a local inflammatory site. In conclusion, PMN are essential in the pathogenesis of HUS and neutrophilia is not merely an epiphenomenon, but contributes to Stx2-damaging mechanism by potentiating Stx2 toxicity.

Activación de la respuesta inmune innata y específica en los pacientes con sindrome urémico hemolítico: papel dual a traves de la paticipacion en los mecanismos pagogenicos y protectores / Activation of innate and specific immune responses in hemolytic uremic syndrome (HUS)-patients

The central role of the immune system is the preservation of the health against several pathogenic microbes and injury agents. However, on special conditions defensive mechanisms triggered towards the foreign agent can damage the host. Clinical and experimental evidence indicate that inflammatory reaction triggered by the main components of Shiga toxin (Stx)-producing Escherichia coil (STEC), participate in the evolution to the complete form of HUS. When children are diagnosed of HUS, they present evidence that have suffered a very strong and early inflammatory response. These features include: the presence of a marked neutrophilia, the polymorfonuclear leucocytes (PMN) are "deactivated or exhausted" and the monocytes are differentiated towards an inflammatory phenotype (CD14-reduced and CD16-enhanced membrane expression). In addition, HUS-patients show a marked reduction in the absolute and relative number of leucocytes carrying the receptor (CX3CR1) for the chemokine "Fractalkine" (FKN, CX3CL1), which are the classic monocytes and Natural Killer cells (NK). All these cells express a high cytotoxic potencial. The chemokine FKN is expressed in endothelial and epithelial renal cells, and is involved in the pathogenic mechanism of different nephropathies. Noteworthy, we found a significant correlation between the severity of the renal damage (as days of anuria) and the alterations described above. Finally, the protective role of specific immune response, mainly through the antibody production with Stx-neutralizing capacity, is discussed.(AU)

Activación de la respuesta inmune innata y específica en los pacientes con sindrome urémico hemolítico: papel dual a traves de la paticipacion en los mecanismos pagogenicos y protectores / Activation of innate and specific immune responses in hemolytic uremic syndrome (HUS)-patients

The central role of the immune system is the preservation of the health against several pathogenic microbes and injury agents. However, on special conditions defensive mechanisms triggered towards the foreign agent can damage the host. Clinical and experimental evidence indicate that inflammatory reaction triggered by the main components of Shiga toxin (Stx)-producing Escherichia coil (STEC), participate in the evolution to the complete form of HUS. When children are diagnosed of HUS, they present evidence that have suffered a very strong and early inflammatory response. These features include: the presence of a marked neutrophilia, the polymorfonuclear leucocytes (PMN) are "deactivated or exhausted" and the monocytes are differentiated towards an inflammatory phenotype (CD14-reduced and CD16-enhanced membrane expression). In addition, HUS-patients show a marked reduction in the absolute and relative number of leucocytes carrying the receptor (CX3CR1) for the chemokine "Fractalkine" (FKN, CX3CL1), which are the classic monocytes and Natural Killer cells (NK). All these cells express a high cytotoxic potencial. The chemokine FKN is expressed in endothelial and epithelial renal cells, and is involved in the pathogenic mechanism of different nephropathies. Noteworthy, we found a significant correlation between the severity of the renal damage (as days of anuria) and the alterations described above. Finally, the protective role of specific immune response, mainly through the antibody production with Stx-neutralizing capacity, is discussed.

[Stabwound of the cervical spinal cord. Two case reports]. / Plaie médullaire cervicale par arme blanche. Présentation de deux cas.

Two cases of Brown-Sequard syndrome following a stab wound of the cervical spinal cord are reported. Spinal cord hemisection was confirmed by magnetic resonance imaging and surgical exploration. Both patients presented leakage of the cerebrospinal fluid and underwent surgical repair. In the first case, the pia-mater was sutured to close the wound and decrease the risk of post-traumatic syringomyelia. Outcome at ten and two years follow up was good in both patients who were able to walk. One of them returned to work. The contribution of surgical repair of spinal cord stab wounds and mechanisms of recovery are discussed.

Prenatal diagnosis of a craniopharyngioma: a new case with radical surgery and review.

CASE REPORT: A case of the antenatal diagnosis of a craniopharyngioma with radical surgery in the neonatal period is reported. REVIEW OF THE LITERATURE: We have reviewed the literature of such cases in an attempt to isolate specific features in this age group and to determine the appropriate management. Only six cases of the truly antenatal diagnosis of craniopharyngiomas have been reported. Diagnosis has resulted from routine ultrasound during pregnancy or from polyhydramnios. Clinically, there is often macrocephaly due to hydrocephalus or a significant-sized tumor. CONCLUSIONS: Management of these rare cases is controversial with high postoperative mortality and significant morbidity, including panhypopituitarism, visual disturbance, and neuropsychological disorders. From the available literature, no conclusions concerning the management can be drawn at present, due to the rarity of early surgical intervention. Our case, despite the lack of important follow-up, seems to confirm the possibility of attempting radical surgery in the neonatal period as a result of advances both in surgical techniques and in neonatal intensive care.

Endogenous glucocorticoids modulate neutrophil function in a murine model of haemolytic uraemic syndrome.

Haemolytic uraemic syndrome (HUS) is caused by Shiga-toxin-producing Escherichia coli (STEC). Although, Shiga toxin type 2 (Stx2) is responsible for the renal pathogenesis observed in patients, the inflammatory response, including cytokines and polymorphonuclear neutrophils (PMN), plays a key role in the development of HUS. Previously, we demonstrated that Stx2 injection generates an anti-inflammatory reaction characterized by endogenous glucocorticoid (GC) secretion, which attenuates HUS severity in mice. Here, we analysed the effects of Stx2 on the pathogenic function of PMN and the potential role of endogenous GC to limit PMN activation during HUS development in a murine model. For this purpose we assessed the functional activity of isolated PMN after in vivo treatment with Stx2 alone or in simultaneous treatment with Ru486 (GC receptor antagonist). We found that Stx2 increased the generation of reactive oxygen intermediates (ROI) under phobol-myristate-acetate (PMA) stimulation and that the simultaneous treatment with Ru486 strengthened this effect. Conversely, both treatments significantly inhibited in vitro phagocytosis. Furthermore, Stx2 augmented in vitro PMN adhesion to fibrinogen (FGN) and bovine serum albumin (BSA) but not to collagen type I (CTI). Stx2 + Ru486 caused enhanced adhesion to BSA and CTI compared to Stx2. Whereas Stx2 significantly increased migration towards N-formyl-methionyl-leucyl-phenylalanine (fMLP), Stx2 + Ru486 treatment enhanced and accelerated this process. The percentage of apoptotic PMN from Stx2-treated mice was higher compared with controls, but equal to Stx2 + Ru486 treated mice. We conclude that Stx2 activates PMN and that the absence of endogenous GC enhances this activation suggesting that endogenous GC can, at least partially, counteract PMN inflammatory functions.

[Ankylosed spine fractures with spondylitis or diffuse idiopathic skeletal hyperostosis: diagnosis and complications]. / Fractures du rachis sur ankylose étendue par spondylarthrite ou hyperostose vertébrale: diagnostic et complications.

PURPOSE OF THE STUDY: Spinal fractures in patients with ankylosing spondylitis or idiopathic skeletal hyperostosis can raise difficult diagnostic and therapeutic problems. Spinal fracture is well known in ankylosing spondylitis but exceptional in diffuse idiopathic skeletal hyperostosis. The purpose of the present work was to identify clinical and radiological features in patients with ankylosing spondylitis, to determine whether similar risks and clinical expression are observed in patients with diffuse idiopathic skeletal hyperostosis, and to present a radiological classification of these fractures. We did not assess therapeutic methods in the present study. MATERIAL AND METHODS: Forty-eight fractures in 48 patients were observed over a period of 17 years. Twenty patients (mean age 62 years) had ankylosing spondylitis and 28 patients (mean age 81 years) had diffuse idiopathic skeletal hyperostosis. A fall was the immediate cause of the fracture in more than half of the patients. No notion of trauma could be identified in six patients. The radiological classification was established as follows; type I open-wedge anterior fracture, type II "sawtooth" fracture, type III occult or radiologically invisible fracture, type IV non-specific fractures comparable to other spinal fractures. A computed tomography was obtained in all patients seen after 1992 and magnetic resonance imaging was performed in case of suspected extradural hematoma. The ASIA classification (as modified by Frankel) was used for cord injuries. Clinical course and complications were noted. RESULTS: Diagnosis was established the day of fracture in 32 patients (12 spondylitis and 20 hyperostosis) and between day 2 and 30 for 16 (8 spondylitis and 8 diffuse idiopathic skeletal hyperostosis). The radiological classification was: type I n=30, type II n=4, type III n=8, type IV n=6 (one odontoid fracture, five compression fractures). Three patients had extradural hematomas (2 spondylitis and 1 hyperostosis). Thirty-four patients (11 spondylitis and 23 hyperostosis) had cord injuries, including 16 with a symptom-free interval. The ASIA classification was: type A n=4, type B n=6, type C n=20, type D n=4. Thirty-two patients died within the first three months after spinal fracture (10 spondylitis and 22 hyperostosis), due to bed rest related complications in 30. One patient died after rupture of an aortic aneurysm. DISCUSSION: Spinal fractures in patients with ankylosing spondylitis or diffuse idiopathic skeletal hyperostosis generally occur spontaneously or after low-energy trauma. Subsequent complications have serious consequences. Late diagnosis either results from missing a radiologically visible fracture or from the presence of an occult "paper thin" fracture. We do not have experience with diagnostic scintigraphy or magnetic resonance imaging. In our opinion, repeating standard x-rays the second and third weeks and use of a spiral scan or multiple spiral scan could provide early diagnosis. CONCLUSION: The possible diagnosis of spinal fracture should be explored very extensively in patients with a symptomatic ankylosed spine who present symptoms compatible with spinal fracture, with or without trauma.

[Infratentorial localisation of a dysembryoplastic neuroepithelial tumor. A case report]. / Localisation sous-tentorielle d'une tumeur dysembryoplasique neuroépithéliale. A propos d'un cas.

Neuroepithelial dysembryoplastic tumor (DNT) is usually considered as a supratentorial benign neoplasm. DNT of the posterior fossa is a very rare entity and only four previous cases were reported in the literature. We describe a case of a 26-Year-old woman presenting recurrent episodes of vertigo. Magnetic resonance imaging revealed four cystic lesions located in the cerebellum, hypointense on T1-weighted images and hyperintense on T2-weighted images, without gadolinium enhancement. After partial resection, histological examination showed small glial cells, oligodendrocytes-like, lying in an eosinophilic alveolar matrix with some floating neurons. Due to this specific glioneuronal element, the diagnosis of DNT was retained. We discuss the clinical and radiological particularities of this infratentorial location and compare our case with those previously described in the literature.

Endogenous glucocorticoids attenuate Shiga toxin-2-induced toxicity in a mouse model of haemolytic uraemic syndrome.

The concept that during an immune challenge the release of glucocorticoids (GC) provides feedback inhibition on evolving immune responses has been drawn primarily from studies of autoimmune and/or inflammatory processes in animal models. The epidemic form of haemolytic uraemic syndrome (HUS) occurs secondary to infection with Gram-negative bacteria that produce Shiga toxin (Stx). Although Stx binding to the specific receptors present on renal tissue is the primary pathogenic mechanism, inflammatory or immune interactions are necessary for the development of the complete form of HUS. The aim of this study was to investigate the influence of endogenous GC on Stx-toxicity in a mouse model. Stx2 was injected into GC-deprived mice and survival rate, renal damage and serum urea levels were evaluated. Plasma corticosterone and cytosolic GC receptor (GR) concentration were also determined at multiple intervals post-Stx2 treatment. Higher sensitivity to Stx2 was observed in mice lacking endogenous GC, evidenced by an increase in mortality rates, circulating urea levels and renal histological damage. Moreover, Stx2 injection was associated with a transient but significant rise in corticosterone secretion. Interestingly, 24 h after Stx inoculation significant increases in total GR were detected in circulating neutrophils. These results indicate that interactions between the neuroendocrine and immune systems can modulate the level of damage significantly during a bacterial infection.

Analysis of the adjuvant effect of recombinant Leishmania infantum Hsp83 protein as a tool for vaccination.

The properties of Leishmania infantum hsp83 (LiHsp83) to elicit an immune response against a fused reporter antigen, maltose binding protein (MBP), was studied. CF1 mice were immunized with different purified recombinant proteins: MBP, LiHsp83 and MBP fused to LiHsp83 (MBP-LiHsp83). Serum samples were obtained at days 0, 21, 28, 60, 90, 120 and 150 post-immunization. MBP-LiHsp83 fusion protein elicited a strong humoral response against MBP, higher than that one obtained in mice immunized with MBP alone or MBP mixed with LiHsp83, showing the secretion of both anti-MBP IgG2a and IgG1 isotypes (IgG2a/IgG1 ratio: 2:1). This response was specific for recombinant proteins and was maintained for at least 150 days, whereas the reactivity in mice immunized with MBP alone dissapeared at day 90. After in vitro stimulation with MBP, spleen cells from MBP-LiHsp83 immunized mice showed higher proliferation indices and produced higher secretion of IFN-gamma than spleen cells from either control or MBP-immunized mice. In all groups of mice IL-4 was undetectable. Thus we consider that LiHsp83 may be a promising candidate to be used as carrier of fused antigens for adjuvant-free vaccination.

Fibrinogen promotes neutrophil activation and delays apoptosis.

The acute phase of the inflammatory response involves an increase in the concentrations of different plasma proteins that include fibrinogen (Fbg) and multiple proinflammatory mediators. In parallel, neutrophil activation is thought to play a crucial role in several inflammatory conditions, and it has been recently demonstrated that Fbg specifically binds to the alpha-subunit of CD11b/CD18 on neutrophil surface. Although several reports have shown that CD11b engagement modulates neutrophil responses, the effect of human Fbg (hFbg), one of CD11b physiologic ligands, has not been exhaustively investigated. We have now shown that incubation of purified neutrophils with hFbg induces a transient and rapid elevation of free intracellular Ca2+. This early intracellular signal is accompanied by changes in the expression of neutrophil activation markers, including enhancement of CD11b and CD66b, and down-regulation of FcgammaRIII. In addition, we have evaluated the effect of hFbg on two functional events related to expression and resolution of inflammation: cytotoxic capacity and rate of neutrophil apoptosis. We have found that activation of neutrophils by hFbg resulted in both enhancement of phagocytosis and Ab-dependent cellular cytotoxicity, and delay of apoptosis. We conclude that during inflammatory processes, soluble Fbg could influence neutrophil responses, increasing and prolonging their functional capacity.

Shiga toxin-2 induces neutrophilia and neutrophil activation in a murine model of hemolytic uremic syndrome.

It has been demonstrated that infections due to Shiga toxins (Stx) producing Escherichia coli are the main cause of the hemolytic uremic syndrome (HUS). Although it is recognized that Stx damage the glomerular endothelium, clinical and experimental evidence suggests that the inflammatory response is able to potentiate Stx toxicity. Lipopolysaccharides (LPS) and neutrophils (PMN) represent two central components of inflammation during a gram-negative infection. In this regard, patients with high peripheral PMN counts at presentation have a poor prognosis. Since the murine model has been used to study LPS-Stx interactions, we analyzed the effects of Stx alone or in combination with LPS on the kinetics of neutrophil production and activation and their participation in renal damage. We observed a sustained neutrophilia after Stx2 injection. Moreover, these neutrophils showed increased expression of CD11b, enhanced cytotoxic capacity, and greater adhesive properties. Regarding the cooperative effects of LPS on Stx2 action, we demonstrated potentiation of neutrophilia and CD11b induction at early times by pretreatment with LPS. Finally, a positive correlation between neutrophil percentage and renal damage (assayed as plasmatic urea) firmly suggests a role for PMN in the pathogenesis of HUS.

Evidence of genetic heterogeneity in a BALB/c mouse colony as determined by DNA fingerprinting.

A genetic monitoring of the BALB/c mouse foundation colony in our animal facility was carried out. The techniques of choice were skin grafting, coat colour test, flow cytometric analysis for H2 antigens (loci H2-D and H2-A), electrophoretic analysis of isoenzymes (loci Idh1, Pep3, Es3 and Mod1), PCR-amplified microsatellites (loci Igh-V, Ngfg, Plau, Crp, Igh, D16Mit5, D3Mit49 and D17Mit16) and DNA fingerprinting (multilocus probes 33.6, 33.15 and (CAC)5). No evidence of genetic contamination was found, ruling out the possibility of an outcross with AKR, the other albino strain maintained at the facility. Nevertheless, DNA fingerprint patterns revealed evidence of genetic heterogeneity in four out of nine lines of the nucleus colony, interpreted as minisatellite mutations favoured for a single line system with more than 40 generations of separation from the ancestral pair. These mice are mainly used in cancer and immunological research within the institute.

Nitric oxide synthase inhibitors enhance plasma levels of corticosterone and ACTH.

The role of nitric oxide in the regulation of adrenal steroidogenesis was examined in BALB/c mice by employing the nitric oxide synthase inhibitors NG-nitro L-arginine methyl ester (L-NAME) and NG-nitro L-arginine (L-NNA). The administration of a single dose of nitric oxide inhibitors (50 mg kg-1 body wt. i.p.) induced a fourfold increase in plasma corticosterone. Treatment with L-arginine (750 mg kg-1 body wt. s.c.), but not D-arginine, completely prevented corticosterone increases induced by L-NAME. To analyse whether the activation of adrenal steroidogenesis induced by nitric oxide synthase inhibitors involved the stimulation of the hypothalamo-pituitary-adrenal axis. ACTH levels were assessed. It was found that L-NAME significantly enhanced plasma ACTH concentrations. Genetic variations in this regulatory pathway are suggested by the fact that L-NAME increased corticosterone levels in BALB/c. C3H/He and DBA-2 mice, but not in C57BI/c mice, a strain characterized by a low steroid response to stress.

Neutrophil apoptosis induced by proteolytic enzymes.

In this study, we show that three proteolytic enzymes of different specificity-pronase, chymotrypsin, and trypsin-induced a dramatic stimulation of neutrophil apoptosis as shown by morphologic characteristics, analysis of cell DNA content, and presence of a characteristic "ladder" pattern of DNA fragmentation. The action of either chymotrypsin or trypsin was completely prevented by the serine protease inhibitor aprotinin, indicating that the proteolytic activity of the enzymes accounts for apoptosis induction. Stimulation of neutrophil apoptosis by proteases was observed in culture medium supplemented with either inactivated fetal calf serum (0.1-50%), autologous serum (0.1-50%), bovine serum albumin (0.1%), or in protein-free medium. Other cell types such as human peripheral blood monocytes and lymphocytes, human leukemic cells from THP-1, HL-60 and K562 lines, murine L929 fibroblasts, and unstimulated murine macrophages harvested from the peritoneal cavity were not induced to undergo apoptosis after the treatment with proteases. In an attempt to determine whether neutrophil serine proteases could induce apoptosis as chymotrypsin and trypsin do, the effect of elastase was assessed. A significant increase in the percentage of apoptotic cells was observed in elastase-treated neutrophils. We propose that the selective stimulation of neutrophil apoptosis by proteolytic enzymes may play an important role in the normal resolution of inflammation by limiting the autotoxic potential of the neutrophil.

Effect of nitric oxide donors on neutrophil responses induced by immune complexes.

The present study characterizes the effect of two nitric oxide (NO) donors, S-nitrosoglutathione (GSNO) and sodium nitroprusside (SNP), on the ability of neutrophils to perform different responses triggered by immune complexes (IC). Pretreatment of neutrophils with either GSNO or SNP exerted a biphasic action on antibody-dependent cellular cytotoxicity (ADCC) performed against erythrocytes (E) coated with IgG antibodies (IgG-E), depending on the amount of IgG employed. While with high amounts of antibodies ADCC was markedly inhibited, at low amounts of antibodies it was significantly increased. Both effects were prevented by haemoglobin, a NO scavenger. Moreover, these effects were reproduced by the cell-permeable analogue of cGMP, dibutyryl cGMP (Bt2cGMP). Other neutrophil functions triggered by IgG-E were also examined. It was found that NO donors did not affect either the phagocytosis of IgG-E or the emission of chemiluminescence (CL). Finally, neutrophil functions triggered by soluble IC (sIC) and precipitating IC (pIC) were analysed. It was observed that NO donors did not modify either cytotoxicity performed towards non-sensitized target cells or CL emission. The significance of these results is discussed.

Effect of medroxyprogesterone acetate (MPA) and serum factors on cell proliferation in primary cultures of an MPA-induced mammary adenocarcinoma.

The effect of progesterone (Pg), medroxyprogesterone acetate (MPA), estradiol (E2), dihydrotestosterone (DHT) and dexamethasone (DEXA) was studied on the in vitro growth rate of a progestin-dependent (PD), estrogen-sensitive mammary tumor line originated in an MPA-treated BALB/c mouse (C4-HD), and on its estrogen-resistant variant (C4-HDR). The specificity of hormone action was further investigated using the anti-hormones RU-486 and hydroxyflutamide (FLU). Cell growth was evaluated in epithelial and fibroblast-enriched cultures using 3H-thymidine and/or autoradiography and immunocytochemistry. The results indicate that cell growth is directly stimulated by MPA and Pg at concentrations ranging from 10(-11) to 10(-7) M. RU486 prevented MPA-induced stimulation in concentrations 10 to 100 fold lower than those of MPA. When used alone, it inhibited cell proliferation only in concentrations higher than 10(-11) M. At nM concentrations, neither DEXA nor DHT stimulated 3H-thymidine uptake except DEXA at 100 nM. MPA-induced stimulation was not reverted by micromolar concentrations of FLU. As for E2 (10(-7)-10(-9) M) it prevented MPA stimulation only in cultures of estrogen-sensitive tumors. Progesterone receptors (PR) (475 +/- 115 fmoles/10(5) cells, n = 5) and estrogen receptors (ER) (ND-115 fmoles/10(5) cells, n = 5) were detected only in epithelial-enriched cultures. Serum from 7 day-MPA-treated mice induced a significant increase of 3H-thymidine uptake; an increase was also obtained with serum from untreated ovariectomized animals to which 1 nM-100 nM concentrations of MPA had been added. The stimulatory effect of the exogenous MPA was much lower than that of the serum obtained from MPA-treated animals. It is concluded that MPA stimulates cell growth of primary cultures of MPA-induced PD tumors via PR. The results provide support for a direct effect of MPA which may be mediated or potentiated by serum factors.

Effect of sialoadenectomy on medroxyprogesterone-acetate-induced mammary carcinogenesis in BALB/c mice. Correlation between histology and epidermal-growth-factor receptor content.

To evaluate the possible involvement of the salivary glands in the modulation of medroxyprogesterone (MPA)-induced mammary tumorigenesis, 48 sialoadenectomized virgin BALB/c female mice and 47 controls were treated with 40mg MPA depot s.c. every 3 months for 1 year. Mammary tumors developed in 11 sialoadenectomized and in 34 control mice with similar latencies. In both groups, 75% of the tumors were ductal and progestin-dependent (PD) while the remainder were lobular and progestin-independent (PI). Epidermal growth factor (EGF) levels were measured in salivary glands (SG-EGF) and serum (S-EGF) in both groups. MPA induced a significant increase in SG-EGF and in S-EGF that became evident only after 1 month of MPA treatment. No increase in S-EGF was detected in MPA-treated sialoadenectomized mice, indicating that salivary glands are the major source of S-EGF. The presence of EGF receptors (EGF-R) was investigated in ductal PD and PI tumor lines and compared with 8 PI tumor lines of lobular origin. A significant difference in EGF-R content was found between lobular and ductal tumors. No increase in EGF-R was noted when ductal tumors became autonomous. EGF-R did not correlate with tumor growth rate and there was an inverse correlation between EGF-R and steroid receptors. When the effect of sialoadenectomy on tumor growth was tested in vivo in syngeneic transplants of 2 ductal PD, 1 ductal PI and 2 lobular PI mammary adenocarcinomas, it was not found to be significant when compared with the controls. It may be concluded that SG-EGF plays an important role in the induction of mammary adenocarcinomas by MPA, while it has no significant effect on the growth of established tumors.